Clonal strains of pituitary tumor cells will be used to investigate the mechanism of action of thyrotropin-releasing hormone (TRH) and of feedback regulation by thyroid hormones at the pituitary level. A fluorescently labeled, biologically active derivative of TRH will be used to study the subcellular distribution of TRH receptors on tumor cells, to investigate the mechanism of regulation by various drugs, and to determine whether reorganization of receptors on the cell surface occurs. Dispersed viable cells from the pituitary glands of normal animals subjected to a variety of hormonal manipulations will also be studied to determine the fraction of anterior pituitary cells with TRH binding sites under different conditions. Thyroid hormones have been shown to decrease the concentration of TRH receptors and block various responses to TRH in normal pituitary tissue and in pituitary tumor lines. The mechanism of this feedback control and the possible involvement of nuclear thyroid hormone receptors will be further investigated by studying the kinetics, analogs specificity and inhibitor sensitivites of the effects. Purified microtubules from bovine thyroid glands have been prepared. To investigate the role of microtubules in the secretory process, specific interactions between tubulin, microtubule associated proteins and purified membranes from thyroid colloid droplets and exocytotic vesicles will be investigated.